Current demand for efficient gene delivery agents is huge and still growing. Deciphering of the human genome in 2003 and so-called “postgenomic era” that came afterwards is characterized by large scale research in examining individual genes and corresponding proteins both performed by the academic community and biopharmaceutical industry. These activities require quick access to expressed recombinant proteins. Hence there is high and constant demand for efficient techniques allowing large-scale high throughput production of required proteins after gene transfer.
Lately, the insertion of foreign DNA into bacteria for expression of desired protein has become a routine procedure. This technique is also employed by the pharmaceutical industry both for analysis and production of recombinant proteins with therapeutic potential. The best-known example is synthesis of human insulin in E. coli bacteria. However, synthesis of recombinant proteins from higher organisms in bacteria has many limitations. Unlike mammalian (eukaryotic) cells, bacteria lack enzymes and organelles that are responsible for the processing and modification of protein, such as glycosylation, disulfide bond formation, etc. Usually, bacteria cannot fold larger proteins into correct 3D structures that ensure proper biological activity of the protein.
The efficient gene delivery to eukaryotic cells could solve aforementioned problems and could be applied not only for gene delivery, but also in gene therapy. However, transfection of mammalian cells is a much more complicated procedure than the transformation of bacteria.
The ideal gene delivery method in eukaryotic cells should meet three major criteria: (1) it should efficiently bring DNA into the cell's nucleus and release it there, (2) it should protect DNA against degradation by nucleases and other enzymes (3) the method itself should be non-toxic to the host cells.
Viral vectors are effective gene carriers and meet the first two criteria. However, they have some major disadvantages. Usually viruses have to be chemically or physically inactivated in order to eliminate their pathogenic properties. Therefore, the chances of reversion to a pathogenic virus exist, which are often difficult to evaluate. Use of viruses in vivo presents the problem of immunogenicity for animals and humans. Moreover, viral systems are expensive, difficult to use and complicated to handle. In comparison to the other gene carriers, the use of viral vectors requires special equipment in order to ensure safety of applicant and environment. Moreover, the virus envelope has a definitive volume and therefore can deliver limited size DNA.
Nonviral gene delivery systems can overcome most of the obstacles associated with viral vectors. The biggest advantage of nonviral transfection reagents is lower immune response and easier application procedure. Most nonviral gene delivery systems are synthetic materials and can be classified into two major groups: cationic lipids and cationic polymers. In both cases amino groups provide the required positive charge for DNA compactization. Amino groups are also found in naturally occurring transfection agents such as spermine and spermidine. The advantage of amino groups is the ability to abstract protons and gain a positive charge at physiological pH.
Cationic lipids are well-studied DNA carriers. Nonetheless, with the success of polyethyleneimine (PEI) as a transfection reagent, a lot of attention is given to the cationic polymers. Cationic polymers are able to condense DNA into small particles and initiate cellular uptake via endocytosis. However, the transfection activity and toxicity of these polymers varies widely. Cationic polymers can be classified into four major groups:                1) polylysine and its derivatives,        2) chitosan and other sugars possessing amino groups,        3) polyamidoamine dendrimers,        4) polyethyleneimine and its modifications.Polylysine        
Polylysine (PLL) is one of the first cationic polymers that was used as a transfection reagent. Due to it's polypeptide structure, PLL has a biodegradable nature and this property is essential for it's use in vivo. However, PLL exhibits moderate to high toxicity as well as immunogenicity due to it's peptide backbone. Further, its transfection efficiency is relatively low. In addition, PLL complexes with DNA undergo nonspecific binding to cell membrane only in certain cell lines thereby limiting it's use. The pKa value of lysine ε-amino group is about 10 and therefore at physiological pH all these amines are protonated. Positively charged PLL can make DNA compact and efficiently transfer it into the cell. However, the PLL-DNA complex cannot rapidly escape from the endosomes, since PLL amino groups are fully protonated and they do not work as a “proton sponge” and cannot facilitate endosmolysis. PLL transfection efficiency can be improved by using it with endosmolytic agents, such as chloroquine. PLL has been used as a graft copolymer with poly(lactic-co-glycolic) acid (PLGA) that serves as endosmolytic agent. The PLL-PLGA graft copolymer demonstrated high transfection efficiency and low toxicity when compared with PLL alone.
The modification of PLL by conjugating histidine to lysine residues resulted in a polymer with higher transfection efficiency than PLL/chloroquine mixture. The pKa value of imidazole group in histidine is around 6, thus it possesses buffering ability, can abstract protons and facilitate polymer-DNA endosomal escape. Furthermore, this polymer is less toxic than PLL alone because of the optimized charge density. Another promising method for preparing PLL based polymers involves replacement of lysine with amino acid cysteine. It was demonstrated that polymers with cross-linked cysteine residues have higher transfection activity, indicating that DNA release is triggered by reduction of disulfide bonds.
The application of PLL as a transfection agent is known, for example from Midoux et al. U.S. Pat. No. 5,595,897 and Yu et al. WO 02/42426. Further, Pharmaceutical Research, Vol. 17, No. 2, 2000 discloses various derivatives of PLL including a PEG-PLL block copolymer, PLL grafted copolymers and PLL with an attached hydrophilic segment like PEG. Cationic copolymers bearing hydrophilic segments also are mentioned.
International Journal of Pharmaceutics 229 (2001) 1-21 “Gene delivery with synthetic (non viral) carriers” discloses covalent attachment of hydrophilic methoxy polyethylene glycol groups to PLL.
Chitosan
Chitosan is a natural biodegradable polymer that shows very low toxicity in the cells. Chitosan size can vary from 1.2 kDa to 500 kDa and as transfection agent it is the most efficient in the molecular range between 30 and 170 kDa. Chitosan forms positively charged toroidal complexes with DNA that protects DNA against DNase degradation. It was shown that more than 65% of chitosan amino groups should be protonated in order to obtain stable complexes able to attach to the cells in vitro. The overall transfection ability of chitosan is relatively low. Like PLL, chitosan has poor buffering capacity and its endosomal escape is also slow. The transfection efficiency of chitosan can be amplified by adding endosmolytic enzyme. The application of chitosan as a gene delivery agent is known, for example from Rolland et al. U.S. Pat. No. 6,184,037.
Polyamidoamine Dendrimers
Polyamidoamine (PAMAM) dendrimers have a 3D spherical structure and they represent a novel class of cationic polymers that are used as transfection agents. The synthesis of dendrimers can be controlled and the degree of branching is expressed in the generation of the dendrimer. Therefore, the PAMAM dendrimers can be produced with low degree of polydispersity and that is a big advantage over other cationic polymers that generate highly polydisperse particles. The uniform size of PAMAM polymers can offer reproducible gene delivery results and potential for clinical application. Dendrimers have a star-like structure with the primary amines on the surfaces and tertiary amines inside. Primary amines are highly charged and they bind DNA, while tertiary amines can abstract protons in the endosomal compartment that results in swelling of the endosome and release of the DNA into the cytoplasm. The hydrolytic degradation of the PAMAM dendrimers yields fractured structures. It has been shown that fractured dendrimers demonstrate strongly enhanced gene expression over corresponding intact polymer. It is thought that fractured polymers have increased flexibility, which is crucial to the swelling of the endosome. QIAGEN offers two commercial sixth generation PAMAM dendrimers as transfection agents: PolyFect, an intact dendrimer, and SuperFect, a fractured dendrimer. The application of dendrimers for gene delivery is known also from Garnet et al. U.S. Pat. No. 6,413,941 and Tomalia et al. WO9524221.
Polyethyleneimine
Polyethyleneimine (PEI) is the most active and most intensively studied cationic polymer to date. Behr's group was the first to show that PEI can be an effective transfection agent U.S. Pat. No. 6,013,240, WO96/02655. PEI can be obtained in branched or linear forms. Branched PEI is known, for example, from AAPS PharmSci 2002; 4(3) article 12 “Transfection Efficiency and Toxicity of Polyethyleneimine in Differentiated Calu-3 and Nondifferentiated Cos-1 Cell Cultures”.
Since every third atom in the PEI chain is nitrogen with pKa value 5.5, PEI is very densely charged polymer. At physiological pH one sixth of nitrogen atoms are protonated. Branched PEI has a ratio of primary:secondary:tertiary amine groups approximately 1:1:1.
Both linear and branched PEI can be used for transfection in vitro and in vivo. However, it has been reported that linear PEI is a less toxic, more efficient and faster acting transfection agent than branched PEI. This could be attributed to the phenomenon that linear PEI-DNA complexes are less condensed and they can penetrate the cell wall and subsequently the cell nucleus more efficiently than branched PEI and DNA complexes.
PEI is slightly toxic to the cells and this can be explained by its nonbiodegradable nature. Studies have shown that PEI transfection efficiency is dependent on its molecular weight. The most active are: 25 kDa branched PEI and 22 kDa linear PEI. Longer linear PEI also show similar transfection activity, but they are more toxic. On the contrary, shorter linear PEI is less toxic and less efficient.
It is believed that highly cationic PEI condenses DNA molecules into small particles and facilitates cellular uptake via endocytosis by interacting with negatively charged cell surface sites. Furthermore, PEI has a big buffering capacity and it can act as “a proton sponge” and can ensure endosomal escape.
Several different strategies have been examined in order to increase transfection activity of PEI. AAPS Journal 2007; 9(1) article 9 “Nonviral Gene Delivery: What We Know and What is Next” discloses coupling of low molecular weight PEI with bifunctional cross-linking reagents bearing biodegradable bonds such as disulfide or ester. This resulted in polymers that are as efficient transfection reagents as 25 kDa PEI, but less toxic to the cells. The purpose of the cross-linking relates to the biodegradability of the polymer, since disulfide and ester bonds can be cleaved within the cells. “Efficient gene transfer using reversibly cross-linked low molecular weight polyethylenimine” Bioconjug Chem. 2001; 12: 989-994 discloses PEI crosslinked with dithiobis (succininimidylpropionate) (DSP) or dimethyl.3,3′-dithiobispropionimidate.2HCl (DTBP). “A degradable polyethylenimine derivative with low toxicity for highly efficient gene delivery” Bioconjug Chem. 2003, 14, 934-940 discloses PEI with small diacrylate cross-linkers.
Linear PEI is a commercialized transfection reagent, known in the market as jetPEI and Exgen500.
The linear PEI is used extensively in vivo. It has been used for gene delivery directly to various anatomical sites of experimental animals as well as introduced systemically by intravenous injection. Experiments have shown that linear PEI polyplexes with DNA are superior to cationic liposomes for gene delivery by intravenous and intratracheal administration. (Bragonzi, A., Boletta, A., Biffi, A., Muggia, A., Sersale, G., Cheng, S. H., Bordignon, C., Assael, B. M., Conese, M., 1999. Comparison between cationic polymers and lipids inmediating systemic gene delivery to the lungs. Gene Ther. 6, 1995-2004; Ferrari, S., Moro, E., Pettenazzo, A., Behr, J. P., Zacchello, F., Scarpa, M., 1997. ExGen 500 is an efficient vector for gene delivery to lung epithelial cells in vitro and in vivo. Gene Ther. 4, 1100-1106.)
PEI with PEG graft copolymer is known from AAPS Journal 2005; 7(1) article 9 “DNA-based Therapeutics and DNA delivery systems: A Comprehensive Review. The PEG is a different polymer to the PEI polymer.
In view of the above it will be seen that there is a need to provide further, preferably improved, transfection reagents for delivering DNA into cells.